The references cited below are not admitted to be prior art to the inventions described herein.
Asparaginases are enzymes which catalyze the deamidation of L-asparagine (asparaginase activity) and L-glutamine (glutaminase activity). See Cantor, P. S. & Schimmell, M. R., Enzyme Catalysis, 2nd ed., (T. Pettersonn & Y. Tacashi, eds.) Sanders Scientific Press, New York pp. 219-23. (1990). L-glutamine serves as the amide donor in purine biosynthesis, as well as other transamination reactions, and hence plays a role in DNA and cyclic nucleotide metabolism.
In vivo biochemical activity of asparaginase was first documented to be present in guinea pig serum in 1922 (see Clementi, A., La desamidation enzmatique de l'asparagine chez les differentes especes-animals et la signification physiologique de sa presence dass l'organisme, 19 Arch. Intern. Physiol. 369 (1922)). The subsequent discovery that asparaginase isolated from guinea pig serum was the active agent which inhibited the in vivo growth of certain asparagine-dependent mammalian tumors without concomitant deleterious effects on normal tissue (see Broome, J. D., Evidence that the asparaginase activity of guinea pig serum is responsible for its anti-lymphoma effects, 191 Nature 1114 (1961)) suggested that this enzyme could be utilized as an anti-neoplastic agent. Because L-asparagine is a non-essential amino acid, asparaginase was initially thought to represent a unique prototype of selective chemotherapy in which treatment could be directed specifically and selectively against asparagine-dependent cells. However, the low levels of asparaginase in guinea pig serum necessitated the development of a more practical source of this anti-neoplastic enzyme.
Subsequently, microbial asparaginase isolated from Escherichia coli and Erwinia carotovora were shown to act as potent anti-leukemic agents (see Howard, J. B. & Carpenter, F. H., L-asparaginase from Erwinia carotovora: substrate specificity and enzymatic properties, 247 J. Biol. Chem. 1020 (1972); Campbell, H. A., et al., Two asparaginases from Escherichia coli B: their separation, purification, and anti-tumor activity, 6 Biochemistry 721 (1967)), and when one of these enzymes was utilized in combination with the chemotherapeutic agent vincristine and the corticosteroid prednisone for the treatment of acute lymphoblastic or acute undifferentiated human leukemia, an overall remission rate of 93% was reported (see Ortega, J. A., et al., L-asparaginase, vincristine, and prednisone for the induction of first remission in acute lymphocytic leukemia, 37 Cancer Res. 535 (1977)).
While these asparaginases possess potent anti-leukemic activity, clinical utilization of the aforementioned microbial asparaginases resulted in a wide range of host toxicity (e.g., hepatic, renal, splenic, pancreatic dysfunction and blood coagulation) and pronounced immunosuppression (see Ohno, R. & Hersh, E. M., Immunosuppressive effects of L-asparaginase, 30 Cancer Res. 1605 (1970)), unlike asparaginase isolated from guinea pig serum (see Cooney, D. A., et al., L-asparaginase and L-asparagine metabolism, 10 Ann. Rev. Pharmacol. 421 (1970)).
Examination of the effects of E. coli asparaginase treatment on spleen histology and lymphocyte populations revealed a marked reduction in both the size and reactivity of the splenic germinal centers which was concomitantly associated with a marked reduction in the cytoplasmic immunoglobulin-containing cells (B-cell immunoblasts; see Distasio, J. A., et al., Alteration in spleen lymphoid populations associated with specific amino acid depletion during L-asparaginase treatment, 42 Cancer Res. 252 (1982)). Additionally, examination of the lymphocyte sub-population within the spleen revealed that there was a 40% reduction in the percentage of surface immunoglobulin-expressing cells (B-cells) accompanied by an increase in the ratio of Thy-1.2-expressing cells (T-cells), whereas the ratio of Lyt-2 to Lyt-1 cells remained unchanged in comparison to the control animal group. These results supported the hypothesis that glutamine, or glutamine combined with asparagine depletion initially resulting from administration of E. coli asparaginase, caused a marked decrease in spleen lymphocytic cells of the B-cell lineage.
Another important adverse clinical effect associated with traditional microbial asparaginase treatment is hepatic dysfunction (see Schein, P. S., et al., The toxicity of E. coli asparaginase, 29 Cancer Res. 426 (1969)). Patients treated with E. coli asparaginase generally exhibit decreased plasma levels of albumin, antithrombin III, cholesterol, phospholipids, and triglycerides. Other indications of asparaginase-induced hepatic dysfunction and pathology include fatty degenerative changes, delayed bromosulfophthalein clearance, and increased levels of serum glutamic-oxaloacetic transaminase and alkaline phosphatase. Although some investigators have reported that low dosages of E. coli asparaginase result in limited hepatotoxic complications, sensitive indicators of hepatic function in some patients receiving low dosages, however, still reveals significant hepatic disease which may result in life-threatening coagulopathy (see Crowther, D., Asparaginase and human malignant disease, 229 Nature 168 (1971)).
The hepatotoxic effects of microbial asparaginases may be a result of their capability to hydrolyze both asparagine and glutamine. One biochemical difference between E. coli and E. carotovora asparaginases and the enzyme derived from guinea pig is the non-specific amidohydrolase activity associated with the microbial enzymes (see Howard, J. B. & Carpenter, F. H., (1972) supra; Campbell, H. A., et al., (1967) supra). For example, E. coli asparaginase has been shown to possess a 130-fold greater level of glutaminase activity as compared to the activity of Wolinella succinogenes (previously classified as Vibrio succinogenes) asparaginase. As a result, patients treated with the conventional microbial asparaginases show a marked reduction in serum levels of both glutamine and asparagine (see Schrek, R., et al., Effect of L-glutaminase on transformation and DNA synthesis of normal lymphocytes, 48 Acta Haematol. 12 (1972)), which may demonstrate a possible correlation between glutamine deprivation and asparaginase-induced clinical toxicity (see Spiers, A. D. S., et al., L-glutaminase/L-asparaginase: human pharmacology, toxicology, and activity in acute leukemia, 63 Cancer Treat. Rep. 1019 (1979)).
The relative importance of L-glutamine in mammalian intermediary metabolism served to stimulate further research into the possible role of glutamine deprivation in asparaginase-induced immunosuppression. Lymphoid tissue has been shown to have relatively low levels of glutamine synthetase activity (see El-Asmar, F. A. & Greenberg, D. H., Studies on the mechanism of inhibition of tumor growth by glutaminase, 26 Cancer Res. 116 (1966); Hersh, E. M., L-glutaminase: suppression of lymphocyte blastogenic responses in vitro, 172 Science 139 (1971)), suggesting that these tissues may be particularly sensitive to the depletion of exogenous glutamine. In contrast, some investigators have proposed that asparagine depletion alone may be responsible for asparagine-induced immunosuppression (see Baechtel, F. S., et al., The influence of glutamine, its decomposition products, and glutaminase on the transformation of human lymphocytes, 421 Biochem. Biophys. Acta 33 (1976)).
While the immunosuppressive effect of E. coli and E. carotovora asparaginases are well-documented (see Crowther, D., (1971) supra; Schwartz, R. S., Immunosuppression by L-asparaginase, 224 Nature 276 (1969)), the molecular biological basis of these functions have not yet been fully elucidated. The inhibition of lymphocyte blastogenesis by various L-glutamine antagonists (see Hersh, E. M. & Brown, B. W., Inhibition of immune response by glutamine antagonism: effect of azotomycin on lymphocyte blastogenesis, 31 Cancer Res. 834 (1980)) and glutaminase from Escherichia coli (see Hersh, E. M., (1971) supra) tends to be illustrative of a possible role for glutamine depletion in immunosuppression. It has been also demonstrated that inhibition of the lymphoid blastogenic response to phytohemagglutinin (PHA) by E. coli asparaginase can be reversed by the addition of L-glutamine but not by the addition of L-asparagine. See Simberkoff, M. S. & Thomas, L., Reversal by L-glutamine of the inhibition of lymphocyte mitosis caused by E. coli asparaginase, 133 Proc. Soc. Exp. Biol. (N. Y.) 642 (1970). Additionally, a correlation between immunosuppression and the relative amount of glutaminase activity has been suggested by the observation that E. asparaginase is more effective than E. coli asparaginase in suppressing the response of rabbit leukocytes to PHA (see Ashworth, L. A. E. & MacLennan, A. P., Comparison of L-asparaginases from Escherichia coli and Erwinia carotovora as immunosuppressant, 34 Cancer Res. 1353 (1974)). However, the significance of these in vitro studies is somewhat limited because the in vivo fates of asparaginases and the homeostatic control of asparagine and glutamine may result in a modification of the immunosuppressive effects of anti-neoplastic asparaginases.
Another significant problem associated with the use of microbial asparaginases is that patients treated with E. coli and E. carotovora asparaginases frequently develop neutralizing antibodies of the IgG and IgM immunoglobulin class (see, e.g., Cheung, N. & Chau, K., Antibody response to Escherichia coli L-asparaginase: Prognostic significance and clinical utility of antibody measurement, 8 Am. J. Pediatric Hematol. Oncol. 99 (1986); Howard, J. B. & Carpenter, F. H. (1972) supra), which allows an immediate rebound of serum levels of asparagine and glutamine. In an attempt to mitigate both the toxic effects and immunosensitivity associated with the therapeutic utilization of E. coli and E. carotovora asparaginase, a covalently-modified E. coli asparaginase (PEG-asparaginase) was initially developed for use in patients who have developed a delayed-type hypersensitivity to preparations "native" of E. coli asparaginase (see Gao, S. & Zhao, G., Chemical modification of enzyme molecules to improve their characteristics, 613 Ann. NY Acad. Sci. 460 (1990)). However, subsequent studies established that the initial development of an immune response against E. coli asparaginase resulted in an 80% cross-reactivity against the PEG-asparaginase with concomitant adverse pharmacokinetic effects--neutralization of PEG-asparaginase activity and normalization of the plasma levels of L-asparagine and L-glutamine (see Avramis, V. & Periclou, I., Pharmodynamic studies of PEG-asparaginase (PEG-ASNase) in pediatric ALL leukemia patients, Seventh International Congress on Anti-Cancer Treatment, Paris, France (1997)). The development of antibodies directed against E. coli (EC) asparaginase and the modified PEG-asparaginase in patients is associated with neutralization of the enzymatic activity of both the EC and PEG-asparaginases in vivo, thus potentially resulting in an adverse clinical prognosis.
It is the object of this invention to solve the foregoing problems through the provision of a therapeutically effective and immunologically-distinct, alternative form of asparaginase, i.e., W. succinogenes asparaginase or an analog thereof. Such asparaginases and their preparation are described in detail below, and they can be used to treat patients suffering from diseases responded to asparagine deprivation as first line therapy or, alternatively, to treat patients who had previously developed hypersensitization to other microbial asparaginases, e.g., that derived from E. coli, and/or modified forms of non-W. succinogenes asparaginases, e.g., E. coli or E. carotovora asparaginase that has been pegylated.